Tetrad Scoring
Scoring of yeast tetrads after sporulation is a crucial step in yeast strain construction. Because the four spores in a single tetrad are unordered products of a single meiosis, a lot can be gleaned right away from looking at the markers. Typically, you will have performed Tetrad Dissection and Replica Plating after Sporulation of a diploid yeast strain to get to this point. You will want to take pictures of all of your replica plates. These should typically include a and alpha tests (crosses to complementary auxotroph mating type testing strains plated onto minimal media), markers for your deletions (our set is made with KanMX, so G418 plates, and we also often use URA3, so ura- plates), and for us our deletion collections are MET15 lys2 and met15 LYS2 respectively for alpha and a so we also often score for 2:2 segregation on met- and lys- plates. When scoring, you will want to use a pre-formatted tetrad scoring sheet , and a pre-drawn plate layout template, which can be seen in the attatched tetrad plate labeling convention image. We score the tetrads for met, lys, KanMX, mating type, and any other markers onto the scoring sheet, being careful that all plate pictures are oriented identically and that all spore number and letters are assigned by the same convention each time (see attached labeling template). In the scoring sheet, + indicates growth an that medium and - indicates lack of growth. When in any doubt when looking at something that looks like growth vs. a tiiiiiny bit of growth, consult your local yeast expert. For a single marker (sporulating from the het dip collection), you should get all tetrads 2:2 for the marker, and all + spores should be the deletion. For a two-marker cross (ex: sgf73::URA3 X rds1::KanMX), you should see each marker segregate INDEPENDENTLY 2:2, and any spores that are + for both markers should be ok. For a single-marker two deletion cross, (ex: ubp8::KanMX X ptr2::KanMX), shit just got real, yeast genetics style. Your tetrads will not all be 2:2 for the marker, BUT ONLY the 2:2 tetrads have the double deletion, and either of the + spores should be good to go. The why of this is very cool and will now be glossed over. Because your tetrads are not all 2:2 for the marker, you can't see that your tetrads are "real" tetrads vs. 4 random spores by looking at your deletion marker, which is where your a/alpha, and possibly met and lys plates (if the parents were +/- for met and or lys) should come in handy. These should segregate 2:2 for genuine tetrads, and a/alpha's always got your back for a normal cross for determining real tetrads via 2:2 segregation. All that being said, there are still lots of ways you can get mixed up at this point, which is why you should PCR genotype your strains. Because we use a quick and easy Colony PCR protocol, we genotype the entire tetrad for any spore colony we're going to freeze down from. If your band sizes for all spores in the tetrad for all deletions of interest correspond perfectly, pick from the selective plates for your deletion markers as an additional insurance against picking the wrong spore, and you're good to go. ALMOST! What you should actually do at this point is freeze down the strains, ideally two a and two alpha for the deletions of interest, and then go back a week later, streak out from your frozen stocks, and replica plate the streaks to see that they have the expected growth on replica plates, including mating type. Then, and only then, you can check the final box in your strain construction spreadsheet and start doing experiments.